Synergy between the phage endolysin LysH5 and nisin to kill Staphylococcus aureus in pasteurized milk

Phage-encoded endolysins are recently considered as new biocontrol tools to inhibit pathogens in food. In this work, we have studied the ionic requirements for optimal lytic activity of LysH5, the endolysin encoded by the staphylococcal bacteriophage phi-SauS-IPLA88. LysH5 activity was inhibited by the presence of Mn++ and Zn++ and enhanced by Ca++, Mg++ and NaCl. When LysH5 was combined with nisin, a bacteriocin currently used as a biopreservative in food, a strong synergistic effect was observed. The Minimum Inhibitory Concentrations of nisin and LysH5 were reduced 64- and 16-fold, respectively, as determined in checkerboard microtitre tests. In addition, nisin enhanced 8-fold the lytic activity of LysH5 on cell suspensions. The synergy observed in vitro was confirmed in challenge assays in pasteurized milk contaminated with S. aureus Sa9. Clearance of the pathogen was only achieved by the combined activity of both antimicrobials. As far as we know, this is the first study that exploits the possibilities of hurdle technology combining a phage-encoded endolysin and the bacteriocin nisin for efficient S. aureus inhibition in milk.